From its inception this grant has pioneered the use of defined model systems to investigate the mechanism and macromolecular determinants of nucleosomal array and linker histone-containing chromatin fiber condensation in vitro. In recent years we have come to focus on the functions of the core histone N-terminal tail domains, the nucleosome surface, and the functions of the linker histone (e.g., H1) C-terminal domain in chromatin condensation. A long term goal of the grant has always been to understand how the conformational transitions of chromatin fibers affect nuclear functions such as transcription. Through the use of recombinant histones, we are currently positioned to perform many novel and innovative mutagenesis-based experiments that address how the eukaryotic genome is packaged and organized, and how chromatin fiber architecture is linked to regulation of transcription. To continue to advance this unique and productive research program, over the next funding interval I propose to use model recombinant nucleosomal arrays and linker histone-containing chromatin fibers to: (1) characterize the condensation of rationally designed core histone N-terminal "tail domain" mutants, and analyze the contributions of the nucleosome surface to nucleosomal array condensation, (2) dissect how linker histones function as nucleosome binding and chromatin architectural proteins, by studying the functional role of the linker histone N-terminal domain and by determining how intrinsic protein disorder and specific amino acid composition influence linker histone C-terminal domain function, and (3) establish structure/function relationships involving chromatin architecture and transcription by correlating nucleosomal array and chromatin fiber condensation with their effects on RNA polymerase II-dependent transcription in a novel in vitro model system. The proposed research is innovative, both in terms of the questions being asked, and the magnitude and scale of the assemblages that will be studied in purely recombinant systems (e.g., the nucleosome exceeds 250 kDa, and oligomeric chromatin fibers can have a mass of hundreds of megadaltons). The mutagenesis experiments are driven by numerous specific hypotheses. All of the proposed studies are supported by published or preliminary data, and yet ask entirely new questions. Taken together, the proposed experiments will yield unprecedented insight into the molecular mechanisms that control condensation and decondensation of the genome, and how they are linked to regulation of functions such as transcription. PUBLIC HEALTH RELEVANCE: The genomes of eukaryotic organisms are packaged into chromatin - a repetitive complex of DNA and histone proteins. Chromatin is simultaneously responsible for condensing chromosomal DNA to fit into the nucleus and regulating function such as transcription. The proposed experiments will use in vitro chromatin model systems to greatly increase our understanding of the molecular factors that control genome architecture, and in turn how genome architecture influences transcription by RNA polymerase II.